APPLICATION OF RT-PCR FOR INDEXING AVOCADO SUNBLOTCH VIROID

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse trans cription-polymerase chain reaction (RT-PCR) was developed using ASBVd- specific primers. Amplified cDNA products were analyzed by electrophor esis on nondenaturing 6% polyacrylamide slab gels. The size of the maj or RT-PCR product from ASBVd-infected tissue was estimated to be 250 b p. This product was absent from amplified extracts of uninfected tissu e. The amplification product from ASBVd was sequenced by the dideoxynu cleotide chain termination method, and the sequence was over 97% ident ical to the published sequence. The RT-PCR assay is sensitive enough t o allow viroid detection without requiring large amounts of tissue, hi ghly purified ASBVd, or molecular hybridization.